Neuregulins (NRGs) and NRG receptors comprise a growth factor-receptor tyrosine kinase system for cell-cell signaling that is involved in organogenesis in nerve, muscle, epithelia, and other tissues (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996; Burden et al., Neuron 18:847-855, 1997). The NRG family consists of three genes that encode numerous ligands containing epidermal growth factor (EGF)-like, immunoglobulin (Ig), and other recognizable domains. Numerous secreted and membrane-attached isoforms function as ligands in this signaling system. The receptors for NRGs are all members of the EGF receptor (EGFR) family, and include EGFR (or ErbB1), ErbB2, ErbB3, and ErbB4, also known as HER1 through HER4, respectively, in humans (Meyer et al., Development 124:3575-3586, 1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90: 1867-71, 1993; Marchionni et al., Nature 362:312-8, 1993; Chen et al., J. Comp. Neurol. 349:389-400, 1994; Corfas et al., Neuron 14:103-115, 1995; Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; and Pinkas-Kramarski et al., Oncogene 15:2803-2815, 1997).
The three NRG genes, Nrg-1, Nrg-2, and Nrg-3, map to distinct chromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA 91:9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang et al., Nature 387:509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci. USA 94:9562-9567, 1997), and collectively encode a diverse array of NRG proteins. The most thoroughly studied to date are the gene products of Nrg-1, which comprise a group of approximately 15 distinct structurally-related isoforms (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Peles and Yarden, BioEssays 15:815-824, 1993). The first-identified isoforms of NRG-1 included Neu Differentiation Factor (NDF; Peles et al., Cell 69, 205-216, 1992 and Wen et al., Cell 69, 559-572, 1992), Heregulin (HRG; Holmes et al., Science 256:1205-1210, 1992), Acetylcholine Receptor Inducing Activity (ARIA; Falls et al., Cell 72:801-815, 1993), and the glial growth factors GGF1, GGF2, and GGF3 (Marchionni et al. Nature 362:312-8, 1993).
The Nrg-2 gene was identified by homology cloning (Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; and Higashiyama et al., J. Biochem. 122:675-680, 1997) and through genomic approaches (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). NRG-2 cDNAs are also known as Neural- and Thymus-Derived Activator of ErbB Kinases (NTAK; Genbank Accession No. AB005060), Divergent of Neuregulin (Don-1), and Cerebellum-Derived Growth Factor (CDGF; PCT application WO 97/09425). Experimental evidence shows that cells expressing ErbB4 or the ErbB2/ErbB4 combination are likely to show a particularly robust response to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol. 18:6090-6101, 1998). The Nrg-3 gene product (Zhang et al., supra) is also known to bind and activate ErbB4 receptors (Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998).
An EGF-like domain is present at the core of all forms of NRGs, and is required for binding and activating ErbB receptors. Deduced amino acid sequences of the EGF-like domains encoded in the three genes are approximately 30-40% identical (pairwise comparisons). Moreover, there appear to be at least two sub-forms of EGF-like domains in NRG-1 and NRG-2, which may confer different bioactivities and tissue-specific potencies.
Cellular responses to NRGs are mediated through the NRG receptor tyrosine kinases EGFR, ErbB2, ErbB3, and ErbB4 of the epidermal growth factor receptor family (Busfield et al., 1997, Mol Cell Biol. 17:4007-14; Carraway et al., 1997, Nature 387:512-6; Chang et al., 1997, Nature 387:509-12). High-affinity binding of all NRGs is mediated principally via either ErbB3 or ErbB4 (Ferguson et al., 2000, EMBO J. 19:4632-43). Binding of NRG ligands leads to dimerization with other ErbB subunits and transactivation by phosphorylation on specific tyrosine residues (Honegger et al., 1990, Mol Cell Biol. 10:4035-44; Lemmon and Schlessinger, 1994, Trends Biochem Sci. 19:459-63; Heldin, 1995, Cell. 80:213-23; Hubbard et al., 1998, J Biol Chem. 273:11987-90). In certain experimental settings, nearly all combinations of ErbB receptors appear to be capable of forming dimers in response to the binding of NRG-1 isoforms. ErbB2, however, appears to be a preferred dimerization partner that may play an important role in stabilizing the ligand-receptor complex.
GGF2 has been shown to promote proliferation, differentiation and protection of Schwann cells (Goodearl et al., 1993, J Biol Chem. 268:18095-102; Minghetti et al., 1996 J Neurosci Res. 43:684-93). Expression of NRG-1, ErbB2, and ErbB4 is also necessary for trabeculation of the ventricular myocardium during mouse development (Meyer and Birchmeier 1995, Nature 378:386-90; Gassmann et al., 1995, Nature 378:390-4; Kramer et al., 1996, Proc Nati Acad Sci USA 93:4833-8). GGF2 has also been shown to promote proliferation and protection of cardiomyocyte cells (Zhao et al., 1998, J Biol Chem 273:10261-10269). GGF2-mediated neuroprotection has also been demonstrated in animal models of stroke, although parameters relating to dosing remain undefined.
The present invention advances the use of GGF2 with respect to therapeutic applications by presenting guidance as to methods for GGF2 administration that optimize therapeutic benefit, while limiting adverse effects. The present invention defines target therapeutic windows for GGF2 serum concentration levels that are specified with respect to particular disease conditions.